Mitogen-activated protein kinase (MAP kinase) was first identified as a protein phosphorylase which is activated when a growth factor is added to cultured cells (Proc. Natl. Acad. Sci. USA, 84, 1502-1506, 1987). However, subsequent research revealed that this enzyme is involved in various vital phenomena such as neuronal differentiation (J. Biol. Chem., 265, 4730-4735, 1990), activation of immune cells (J. Immunol., 144, 2683-2689, 1990) and secretions (J. Cell Biol., 110, 731-742, 1990). As regards human MAP kinase, cloning of its gene showed the existence of several molecular species of high homology but the main species are two, namely ERK1 and ERK2. These two proteins are highly homologous (84.7%) (FEBS LETT., 304, 170-178, 1992). Though their functional dissimilarity had been presumed for some time, no difference in vitro, whether in function or in activity, has been discovered to this day.
Several kinds of antibodies against this MAP kinase have been obtained but the majority of them are polyclonal antibodies. In this connection, several monoclonal antibodies are known. Among them are Clone MK12 [Julian Gomez-Cambronero et al., Proc. Natl. Acad. Sci. USA, 89, 7551-7555, 1992] or [Seikagaku Kogyo Catalog (1993/94), p.184] and Clone B9 [UBI Catalog (1993), p.33]. However, there is no report on monoclonal antibodies that would ever distinguish between ERK1 and ERK2. There is not a case, either, in which a monoclonal antibody was ever obtained by immunizing an animal other than man (e.g. mice) with human MAP kinase.
So far, as antibodies capable of distinguishing between ERK1 and ERK2, Simon J. Cook and coworkers have reported polyclonal antibodies specifically binding to these proteins, respectively [EMBO Journal, 12, 3475-3485, 1993] but these antibodies were invariably used in Western blotting and immunoprecipitation only and quantitation of MAP kinase protein has not been done with them.
MAP kinase is a serine-threonine kinase which is activated when both the Thr and Tyr residues in the Thr-Glu-Tyr sequence are phosphorylated and phosphorylation of these two residues are considered to be necessary and sufficient conditions for activation [Neil G. Anderson et al., Nature, 343, 651-653, 1990]. The antibody reported by Ito et al. [Proceedings of 1994 Congress of Japanese Biochemical Society, Lecture No. 4887] and the New England Biolabs antibody are known as antibodies that specifically bind to phosphorylated MAP kinase. However, these are used only in the Western blotting and immunoprecipitation of active-form MAP kinase and immune staining of cells.
No quantitative determination of active-form MAP kinase has been attempted.
So far, as methods for assay of MAP kinase activity, the technique comprising phosphorylating myelin basic protein (MBP) with .gamma.-.sup.32 P-ATP and measuring the radioactivity taken up in the MBP [Ahn, N. et al., J. Biol. Chem., 266, 4220-4227, 1991] and the technique comprising subjecting a cell extract to SDS-PAGE and phosphorylating MBP within the gel [Leevers, S. J. et al., EMBO Journal, 11, 569-574, 1992] are mainly used in practice but these techniques have the drawback that a radioisotope must be employed. As a comparatively expedient method, a technique utilizing the difference in electrophoretic mobility between the active-form and the inactive-form is known [e.g. Johan Van Lint et al., Molecular and Cellular Biochemistry, 127/128, 171-178, 1993] but assays can hardly be done by this technique.